Transposase polypeptides and uses thereof

ABSTRACT

Transposase polypeptides and polynucleotides are provided, which have a high activity in mammalian cells. Methods for engineering cells, such as chimeric antigen T-cells, with the transposes are also provided.

This application is a continuation of U.S. application Ser. No.15/556,868, filed Sep. 8, 2017, as a national phase application under 35U.S.C. § 371 of International Application No. PCT/US2016/021693, filedMar. 10, 2016, which claims the benefit of U.S. Provisional ApplicationNo. 62/131,827, filed on Mar. 11, 2015, the entire contents of each ofwhich is incorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“UTSCP1256USC1_ST25.txt”, which is 18 KB (as measured in MicrosoftWindows®) and was created on Jan. 13, 2020, is filed herewith byelectronic submission and is incorporated by reference herein.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the fields of medicine,immunology, cell biology, and molecular biology. In certain aspects, thefield of the invention concerns transposase polypeptides and the usedthereof in genetic engineering.

2. Description of Related Art

In the era of functional genomics, there is a need for efficient meansto alter the coding sequence in the genome of cells. Such genomeengineering can be used to produce cells with stably expressedtransgenes and for cell reprogramming. One suitable tool used in genomeengineering is transposon/transposase systems. Transposons ortransposable elements include a (short) nucleic acid sequence withterminal repeat sequences upstream and downstream thereof and encodeenzymes that facilitate the excision and insertion of the nucleic acidinto target DNA sequences. Several transposon/transposase systems havebeen adapted for genetic insertions of heterologous DNA sequences,including Sleeping Beauty (SB), a Tc1/mariner-like element from fishthat exhibits transpositional activity in a variety of vertebratecultured cell lines, embryonic stem cells and in vivo (Ivies et al.,1997). However, none of these systems has been adopted forhuman/mammalian cells engineering. Accordingly, there is need fortransposon/transposase systems with a high level of activity inmammalian cells and organisms.

SUMMARY OF THE INVENTION

In a first embodiment is provided a recombinant polypeptide comprisingor consisting of enhanced transposase activity in mammalian cells. Insome aspects, a polypeptide of the embodiments comprises or consists ofa sequence at least 90% identical to SEQ ID NO: 1 (hSB110) or SEQ ID NO:3 (hSB81). In certain aspects, a polypeptide comprises or consists of asequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a sequence at least 90%identical to the full length of SEQ ID NO: 1 or SEQ ID NO:3 and exhibitstransposase activity in mammalian cells. In a further aspect, thepolypeptide comprises or consists of a sequence at least 90% identicalto SEQ ID NO: 1 or SEQ ID NO:3, exhibits transposase activity inmammalian cells, and comprises one or more of the following features: anArg at the position corresponding to position 136 (in hSB110), a His atthe position corresponding to position 253 (in hSB110), an Arg at theposition corresponding to position 255 (in hSB110) and/or a Thr at theposition corresponding to position 314 (in hSB110). In further aspects,the preceding at least 90% identical polypeptide does not comprise thesequence of a naturally occurring transposase enzyme or does notcomprise the sequence of SEQ ID NO: 5 (SB11), SEQ ID NO: 6 (SB10) or SEQID NO: 7 (SB100x). In some aspects, a polypeptide of the embodimentscomprises or consists of a sequence at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 1 (hSB110) or SEQ ID NO: 3(hSB81). In some aspects, a polypeptide of the embodiments comprises orconsists of a sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% identical to SEQ ID NO: 1 (hSB110) or SEQ ID NO: 3 (hSB81) andexhibits transposase activity in mammalian cells. In some aspects, apolypeptide of the embodiments comprises or consists of a sequence atleast 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ IDNO: 1 (hSB110) or SEQ ID NO: 3 (hSB81), exhibits transposase activity inmammalian cells, and comprises or consists of one or more of thefollowing features: an Arg at the position corresponding to position 136(in hSB110), a His at the position corresponding to position 253 (inhSB110), an Arg at the position corresponding to position 255 (inhSB110) and/or a Thr at the position corresponding to position 314 (inhSB110). In some aspects, the preceding at least 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical polypeptide does not comprise thesequence of a naturally occurring transposase enzyme or does notcomprise the sequence of SEQ ID NO: 5 (SB11), SEQ ID NO: 6 (SB10) or SEQID NO: 7 (SB100x). In yet further aspects, a transposase polypeptide ofthe embodiments further comprises or consists of a heterologouspolypeptide sequence fused to the N- or C-terminus of the transposasesequence. For example, the heterologous polypeptide sequence maycomprise or consist of a reporter, a purification tag or a cellpenetrating polypeptide (CPP). In further aspects, the mammalian cell inwhich polypeptides of the embodiments exhibit transposase activity arehuman cells. In further aspects, the human cells are immune cells. Infurther aspects the human immune cells are T cells. In further aspects,the T cells are T helper cells (T_(H) cells), cytotoxic T cells (T_(c)cells or CTLs), memory T cells (T_(CM) cells), effector T cells (T_(EM)cells), regulatory T cells (Treg cells; also known as suppressor Tcells), natural killer T cells (NKT cells), mucosal associated invariantT cells, alpha-beta T cells (Tαβ cells), and/or gamma-delta T cells (Tγδcells).

In yet a further aspect, a polypeptide of the embodiments comprises orconsists of a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to SEQ ID NO: 1 (hSB110) or SEQ ID NO: 3 (hSB81),comprises one or more of the following features: an Arg at the positioncorresponding to position 136 (in hSB110), a His at the positioncorresponding to position 253 (in hSB110), an Arg at the positioncorresponding to position 255 (in hSB110) and/or a Thr at the positioncorresponding to position 314 (in hSB110) and further comprises one ormore of the following additional features: an Arg at the positioncorresponding to position 14 (in hSB110), an Ala at the positioncorresponding to position 33 (in hSB110), a His at the positioncorresponding to position 115 (in hSB110), an Asp at the positioncorresponding to position 214 (in hSB110), an Ala at the positioncorresponding to position 215 (in hSB110), a Val at the positioncorresponding to position 216 (in hSB110), a Gln at the positioncorresponding to position 217 (in hSB110), and/or a His at the positioncorresponding to position 243 (in hSB110). In some aspects, thepreceding at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical polypeptide does not comprise the sequence of a naturallyoccurring transposase enzyme or does not comprise the sequence of SEQ IDNO: 5 (SB11), SEQ ID NO: 6 (SB10) or SEQ ID NO: 7 (SB100x). In someaspects, a polypeptide of the embodiments comprises 2, 3, 4, 5, 6, 7 or8 of the sequence features selected from the group consisting of: an Argat the position corresponding to position 14 (in hSB110), an Ala at theposition corresponding to position 33 (in hSB110), a His at the positioncorresponding to position 115 (in hSB110), an Asp at the positioncorresponding to position 214 (in hSB110), an Ala at the positioncorresponding to position 215 (in hSB110), a Val at the positioncorresponding to position 216 (in hSB110), a Gln at the positioncorresponding to position 217 (in hSB110), and a His at the positioncorresponding to position 243 (in hSB110). In further aspects, thepreceding at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identical polypeptide comprises the sequence DAVQ at the positionscorresponding to positions 214-217(in hSB110).

In some aspects, the polypeptide of the embodiments comprises orconsists of a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to SEQ ID NO: 1 (hSB110), comprises a Asn at theposition corresponding to position 314 (in hSB110) and comprises one ormore of the following features: an Arg at the position corresponding toposition 136 (in hSB110), a His at the position corresponding toposition 253 (in hSB110), and/or an Arg at the position corresponding toposition 255 (in hSB110). In some aspects, the preceding at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical polypeptide doesnot comprise the sequence of a naturally occurring transposase enzyme ordoes not comprise the sequence of SEQ ID NO: 5 (SB11), SEQ ID NO: 6(SB10) or SEQ ID NO: 7 (SB100x). In specific aspects, the polypeptidecomprises or consists of the sequence of SEQ ID NO: 1.

In other aspects, the polypeptide of the embodiments comprises orconsists of a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to SEQ ID NO: 3 (hSB81), comprises a Thr at theposition corresponding to position 314 (in hSB110) and comprises one ormore of the following features: an Arg at the position corresponding toposition 136 (in hSB110), a His at the position corresponding toposition 253 (in hSB110), and/or an Arg at the position corresponding toposition 255 (in hSB110). In some aspects, the preceding at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical polypeptide doesnot comprise the sequence of a naturally occurring transposase enzyme ordoes not comprise the sequence of SEQ ID NO: 5 (SB11), SEQ ID NO: 6(SB10) or SEQ ID NO: 7 (SB100x). In specific aspects, the polypeptidecomprises or consists of the sequence of SEQ ID NO: 3.

In a further embodiment there is provided a polynucleotide moleculecomprising or consisting of a sequence encoding a polypeptide accordingto the embodiments. The molecule may be a DNA expression vector in someaspects. For example, the DNA expression vector may comprise atransposase coding sequence operably linked to a promoter for in vitroexpression of the polypeptide (e.g., a T7 or SP6 promoter) or a promoterfor expression of the polypeptide in mammalian cells. In some aspects,the polynucleotide molecule may be a RNA or mRNA. In further aspects,the RNA may comprise a 5′-cap, an IRES motif, a (heterologous) 5′ UTR, a(heterologous) 3′ UTR and/or a poly(A) sequence. The RNA mayadditionally comprise a poly(A) sequence of 20 to 300 nucleotides insome aspects.

In yet a further embodiment the invention provides a method of making atransposase polypeptide as described above, comprising transfecting acell with a polynucleotide encoding a transposase polypeptide andexpressing the polypeptide from the polynucleotide. In still a furtherembodiment, the invention provides a host cell comprising a polypeptideor a polynucleotide molecule of the embodiments. In some cases, the cellis a mammalian cell, such as a human cell. In certain aspects, the cellis a stem cell or an induced pluripotent stem (iPS) cell. In furtheraspects, the cell is a natural killer (NK) cell, a precursor of a NKcell, a T-cell, a precursor of a T-cell, or an immune cell. In somecases, the cell may comprise a RNA encoding a transposase polypeptide ofthe embodiments. In yet further embodiments a population of cells, saidcells comprising polypeptide or a polynucleotide molecule of theembodiments, is provided.

In another embodiment, a method is provided for genetically engineeringa cell comprising: transfecting the cell with a transposase polypeptideas described above or a nucleic acid encoding the transposasepolypeptide and a DNA vector comprising a sequence encoding a selectedgenetic element flanked by transposon repeats, then incubating the cellunder conditions appropriate for (transient or stable) transposaseactivity, thereby integrating the selected genetic element in the genomeof the cell and producing an engineered cell. In certain aspects, a DNAvector encoding a selected genetic element flanked by transposon repeatsfurther comprises a sequence encoding a transposase polypeptide of theembodiments. Thus, in certain aspects, a method of the embodimentscomprises transfecting a cell with a DNA vector comprising a sequenceencoding a selected genetic element flanked by transposon repeats and asequence encoding a transposase of the embodiments, which is under thecontrol of a promoter sequence, then incubating the cell underconditions appropriate for transposase expression and activity, therebyintegrating the selected genetic element in the genome of the cell andproducing an engineered cell. In some aspects, the method comprises thethird step of isolating or culturing the engineered cell. In someaspects, the selected genetic element is a screenable or selectablemarker. In further aspects, the selected genetic element may encode anantibody, a inhibitory nucleic acid (e.g., a small interfering RNA(siRNA)), a therapeutic polypeptide, a T-cell receptor (TCR), a chimericantigen receptor (CAR), or an enhancer of immune cell function. Inspecific aspects, the selected genetic element encodes a CAR or TCR. Instill further aspects, the selected genetic element may be a gene or aportion thereof that is used to replace or modify the corresponding genefrom a cell (e.g., to alter the sequence or expression of the gene or to“knock-out” gene expression in the cell). In some aspects, thetransfected cell is a mammalian cell such as human cell. In some cases,the cell may be a stem cell or an iPS cell. In certain aspects, the cellmay be an immune system cell or a precursor thereof, such as a NK cell,a T-cell, a precursor of a NK cell, or a precursor of a T-cell.

In some aspects, transfecting cells may comprise use of a chemical-basedtransfection reagent, electroporation of the cells or other technologiesproviding delivery of nucleic acid and/or protein to the cytoplasm andnucleus of cells. For example, cells can be transfected using saltprecipitates (e.g., CaPO₄ precipitates), lipids (e.g., charged ornon-polar lipids), cationic polymers, PEG-complexes and/or proteincomplexes (e.g., cationic polypeptides). In some aspects, transfectionsmay involve the use of liposomes, such as phospholipid liposomes (e.g.,liposomes that incorporate a glycerophospholipid or sphingolipids). Instill further aspects, cells may be transduced with a viral vector(e.g., adenoviral, adeno-associated viral, retroviral (e.g., lentiviral)or vaccinia virus vector). In certain aspects, a viral vector for useaccording to the embodiments is a non-integrating viral vector. Askilled artisan will recognize that, in certain aspects, a transposaseof the embodiments may be delivered to cells together or separately froma nucleic acid encoding transposon repeats. For example, in certainaspects, a transposase may be delivered to cells as a recombinantpolypeptide using a protein transfection reagent and the nucleic acidmolecule comprising the transposon repeats can be delivered using anucleic acid transfection system or a viral vector. In further aspects,an RNA encoding a transposase is co-transfected with a DNA comprisingtransposon repeats and a selected genetic element.

In further aspects, the method additionally comprises transfecting apopulation of cells with a transposase polypeptide of the embodiments ora nucleic acid encoding the transposase polypeptide and a DNA vectorcomprising a sequence encoding a selected genetic element flanked bytransposon repeats and then incubating the population under conditionsappropriate for transposase activity, thereby integrating the selectedgenetic element in the genome of the cells and producing a population ofengineered cells. In specific aspects, the method comprises transfectinga population of T-cells, or T-cell precursors, with a transposasepolypeptide or a nucleic acid encoding the transposase polypeptide and aDNA vector comprising a sequence encoding a CAR flanked by transposonrepeats and incubating the population under conditions appropriate fortransposase activity, thereby integrating the CAR in the genome of thecells and producing a population of engineered T-cells, or T-cellprecursors. In further aspects, the method comprises transfecting apopulation of T-cells, or T-cell precursors, with a DNA vectorcomprising a sequence encoding a CAR flanked by transposon repeats and asequence encoding a transposase of the embodiments (operably linked to apromoter) and incubating the population under conditions appropriate fortransposase activity, thereby integrating the CAR in the genome of thecells and producing a population of engineered T-cells, or T-cellprecursors. Culturing the engineered cells in a medium that selectivelyenhances proliferation of CAR-expressing T-cells may be additionallyperformed in some aspects.

In yet a further embodiment there is provided a method of providing aT-cell response in a human subject having a disease comprising firstobtaining a population of engineered T-cells, or T-cell precursors, inaccordance with the embodiments, optionally culturing the cells in amedium that selectively enhances proliferation of CAR-expressingT-cells, and then administering an effective amount of theCAR-expressing T-cells to the subject to provide a T-cell response.

Thus, in some aspects, a method of the embodiments comprises: (a)obtaining a sample of cells from the subject, the sample comprisingT-cells or T-cell progenitors; (b) transfecting the cells with a DNAencoding a transposon-flanked chimeric antigen receptor (CAR) and atransposase of the embodiments effective to integrate the DNA encodingthe CAR into the genome of the cells, to provide a population oftransgenic CAR-expressing cells; (c) optionally, culturing thepopulation of transgenic CAR cells ex vivo in a medium that selectivelyenhances proliferation of CAR-expressing T-cells; and (d) administeringan effective amount of the transgenic CAR cells to the subject toprovide a T-cell response. Thus, in some aspects, the transgenic CARcells are cultured ex vivo for less than 21 days, such as for less than20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 daysor less. In certain aspects, the CAR cells are cultured ex vivo no morethat 3 to 5 days. In still further aspects, steps (a)-(d) of the instantmethod (i.e., obtaining cell samples to administering CAR T cells) arecompleted in no more than 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, or 5 days. In further aspects, the sample of cells fromthe subject may be a sample of less than about 200 mls of a peripheralblood or umbilical cord blood. In some aspects, the sample may becollected by apheresis. In certain aspects, the sample is collected by amethod that does not involved apheresis (e.g., by venipuncture). Instill further aspects, the sample of cells has an initial volume of lessthan 175 mls, less than about 175 mls, less than 150 mls, less thanabout 150 mls, less than 125 mls, less than about 125 mls, less than 100mls, less than about 100 mls, less than 75 mls, less than about 75 mls,less than 50 mls, less than about 50 mls, less than 25 mls, or less thanabout 25 mls (e.g., the sample of cells has an initial volume of betweenabout 50 and about 200 mls, between about 50 and about 100 mls, orbetween about 100 and about 200 mls when obtained from the subject).

In some aspects, methods of the embodiments concern transfecting thecells with a DNA encoding a chimeric antigen receptor (CAR) and atransposase. Methods of transfecting of cells are well known in the art,but in certain aspects, highly efficient transfections methods such aselectroporation are employed. For example, nucleic acids may beintroduced into cells using a nucleofection apparatus. In certainembodiments, the transfection step does not involve infecting ortransducing the cells with virus, which can cause genotoxicity and/orlead to an immune response against cells containing viral sequences in atreated subject.

Further aspects of the embodiments concern transfecting cells with anexpression vector encoding a CAR. A wide range of CAR constructs andexpression vectors for the same are known in the art. For example, insome aspects, the CAR expression vector is a DNA expression vector suchas a plasmid, linear expression vector or an episome. In some aspects,the vector comprises additional sequences, such as sequence thatfacilitate expression of the CAR, such a promoter, enhancer, poly-Asignal, and/or one or more introns. In certain aspects, the CAR codingsequence is flanked by transposon sequences, such that the presence of atransposase allows the coding sequence to integrate into the genome ofthe transfected cell.

As detailed supra, in certain aspects, cells are further transfectedwith a transposase of the embodiments that facilitates integration of aCAR coding sequence into the genome of the transfected cells. In someaspects, the transposase is provided as DNA expression vector. Incertain aspects, the transposase is provided as an expressible RNA or aprotein such that long-term expression of the transposase does not occurin the transgenic cells. For example, in some aspects, the transposaseis provided as encoded by an mRNA (e.g., an mRNA comprising a cap andpoly-A tail).

In still further aspects, a transgenic CAR cell of the embodimentsfurther comprises an expression vector for expression of amembrane-bound cytokine that stimulates proliferation and/or survival ofT-cells. In particular, CAR cells comprising such cytokines canproliferate and/or persist with little or no ex vivo culture withactivating and propagating cells (AaPCs) or artificial antigenpresenting cells (aAPCs) due to the simulation provided by the cytokineexpression. Likewise, such CAR cells can proliferate in vivo even whenlarge amounts of antigen recognized by the CAR is not present (e.g., asin the case of a cancer patient in remission or a patient with minimalresidual disease). In some aspects, the CAR cells comprise a DNA or RNAexpression vector for expression of a Cγ cytokine and elements (e.g., atransmembrane domain) to provide surface expression of the cytokine. Forexample, the CAR cells can comprise membrane-bound versions of IL-7,IL-15 or IL-21. In some aspects, the cytokine is tethered to themembrane by fusion of the cytokine coding sequence with the receptor forthe cytokine. For example, a cell can comprise a vector for expressionof a IL-15-IL-15Ra fusion protein. In still further aspects, a vectorencoding a membrane-bound Cγ cytokine is a DNA expression vector, suchas vector integrated into the genome of the CAR cells or anextra-chromosomal vector (e.g., and episomal vector). In still furtheraspects, expression of the membrane-bound Cγ cytokine is under thecontrol of an inducible promoter (e.g., a drug inducible promoter) suchthat the expression of the cytokine in the CAR cells (and thereby theproliferation of the CAR cells) can be controlled by inducing orsuppressing promoter activity.

Aspects of the embodiments concern obtaining a sample from a patientcomprising NK cells, NKT cells, T-cells or T-cell progenitor cells. Forexample, in some cases, the sample is an umbilical cord blood sample, aperipheral blood sample (e.g., a mononuclear cell fraction) or a samplefrom the subject comprising pluripotent stem cells. In some aspects, asample from the subject can be cultured to generate induced pluripotentstem (iPS) cells and these cells used to produce NK cells, NKT cells orT-cells. Cell samples may be cultured directly from the subject or maybe cryopreserved prior to use. In some aspects, obtaining a cell samplecomprises collecting a cell sample. In other aspects, the sample isobtained by a third party. In still further aspects, a sample from asubject can be treated to purify or enrich the T-cells or T-cellprogenitors in the sample. For example, the sample can be subjected togradient purification, cell culture selection and/or cell sorting (e.g.,via fluorescence-activated cell sorting (FACS)).

In some aspects, a method of the embodiments further comprises usingantigen presenting cells (e.g., for expansion of engineered cells). Forexample, the antigen presenting cells can be dendritic cells, activatingand propagating cells (AaPCs), or inactivated (e.g., irradiated)artificial antigen presenting cells (aAPCs). Methods for producing suchantigen presenting cells are known in the art and further detailedherein. Thus, in some aspects, transgenic CAR cells are co-cultured withantigen presenting cells (e.g., inactivated aAPCs) ex vivo for a limitedperiod of time in order to expand the CAR cell population. The step ofco-culturing CAR cells can be done in a medium that comprises, forexample, interleukin-21 (IL-21) and/or interleukin-2 (IL-2). In someaspects, the co-culturing is performed at a ratio of CAR cells toantigen presenting cells of about 10:1 to about 1:10; about 3:1 to about1:5; or about 1:1 to about 1:3. For example, the co-culture of CAR cellsand antigen presenting cells can be at a ratio of about 1:1, about 1:2or about 1:3.

In some aspects, cells for culture of CAR cells such as AaPCs or aAPCsare engineered to express specific polypeptide to enhance growth of theCAR cells. For example, the cells can comprise (i) an antigen targetedby the CAR (i.e., that is expressed on the transgenic CAR cells); (ii)CD64; (ii) CD86; (iii) CD137L; and/or (v) membrane-bound IL-15,expressed on the surface of the aAPCs. In some aspects, the AaPCs oraAPCS comprise a CAR-binding antibody or fragment thereof expressed onthe surface of the AaPCs or aAPCs. Preferably, AaPCs or aAPCs for use inthe instant methods are tested for, and confirmed to be free of,infectious material and/or are tested and confirmed to be inactivatedand non-proliferating.

While expansion on AaPCs or aAPCs can increase the number orconcentration of CAR cells in a culture, this procedure is laborintensive and expensive. Moreover, in some aspects, a subject in need oftherapy should be re-infused with transgenic CAR cells in as short atime as possible. Thus, in some aspects, ex vivo culturing thetransgenic CAR cells is for no more than 14 days, no more than 7 days,or no more than 3 days. For example, the ex vivo culture (e.g., culturein the presence of AaPCs or aAPCs) can be performed for less than onepopulation doubling of the transgenic CAR cells. In still furtheraspects, the transgenic cells are not cultured ex vivo in the presenceof AaPCs or aAPCs.

In still further aspects, a method of the embodiments comprises a stepfor enriching the cell population for CAR-expressing T-cells aftertransfection of the cells or after ex vivo expansion of the cells. Forexample, the enrichment step can comprise sorting of the cells (e.g.,via FACS), for example, by using an antigen bound by the CAR or aCAR-binding antibody. In still further aspects, the enrichment stepcomprises depletion of the non-T-cells or depletion of cells that lackCAR expression. For example, CD56⁺ cells can be depleted from a culturepopulation. In yet further aspects, a sample of CAR cells is preserved(or maintained in culture) when the cells are administered to thesubject. For example, a sample may be cryopreserved for later expansionor analysis.

In certain aspects, transgenic CAR cells are inactivated for expressionof an endogenous T-cell receptor and/or endogenous HLA. For example, Tcells can be engineered to eliminate expression of endogenous alpha/betaT-cell receptor (TCR). In specific embodiments, CAR⁺ T cells aregenetically modified to eliminate expression of TCR. In some aspects,there is a disruption of the T-cell receptor α/β in CAR-expressing Tcells using zinc finger nucleases (ZFNs). In certain aspects, the T-cellreceptor α/β-chain in CAR-expressing T cells is knocked-out, forexample, by using zinc finger nucleases.

As further detailed herein, CAR cells of the embodiments can be used totreat a wide range of diseases and conditions. Essentially any diseasethat involves the specific or enhanced expression of a particularantigen can be treated by targeting CAR cells to the antigen. Forexample, autoimmune diseases, infections, and cancers can be treatedwith methods and/or compositions of the invention. These includecancers, such as primary, metastatic, recurrent, sensitive-to-therapy,refractory-to-therapy cancers (e.g., chemo-refractory cancer). Thecancer may be of the blood, lung, brain, colon, prostate, breast, liver,kidney, stomach, cervix, ovary, testes, pituitary gland, esophagus,spleen, skin, bone, and so forth (e.g., B-cell lymphomas or amelanomas). In the case of cancer treatment CAR cells typically target acancer cell antigen (also known as a tumor-associated antigen (TAA)).

In still further aspects, transgenic CAR cells of the embodiments may beused to treat subject having minimal residual disease (e.g., a subjecthaving very low amounts of CAR-targeted antigen present), such as cancerpatients that are in apparent remission. Using new highly sensitivediagnostic techniques, cancer-associated antigens (or cancer cells) canbe detected in patients that do not exhibit overt cancer symptoms. Suchpatients may be treated by the instant methods to eliminate residualdisease by use of antigen-targeted CAR cells. In certain embodiments,transgenic CAR cells for targeting of residual disease further compriseexpression of a membrane-bound proliferative cytokine, as these cellswill retain the ability to expand in vivo despite the low amount oftarget antigen.

The processes of the embodiments can be utilized to manufacture (e.g.,for clinical trials) CAR⁺ T cells with binding specificity for varioustumor antigens (e.g., CD19, ROR1, CD56, EGFR, CD33, CD123, c-met, GD2).CAR⁺ T cells generated using this technology can be used to treatpatients with leukemias (e.g., AML, ALL, CML), infections and/or solidtumors. For example, methods of the embodiments can be used to treatcell proliferative diseases, fungal, viral, bacterial or parasiticinfections. Pathogens that may be targeted include, without limitation,Plasmodium, trypanosome, Aspergillus, Candida, HSV, RSV, EBV, CMV, JCvirus, BK virus, or Ebola pathogens. Further examples of antigens thatcan be targeted by CAR cells of the embodiments include, withoutlimitation, CD19, CD20, carcinoembryonic antigen, alphafetoprotein,CA-125, 5T4, MUC-1, epithelial tumor antigen, melanoma-associatedantigen, mutated p53, mutated ras, HER2/Neu, ERBB2, folate bindingprotein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoproteingp41, GD2, CD123, CD23, CD30, CD56, c-Met, meothelin, GD3, HERV-K,IL-11Ralpha, kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, orVEGFR2. In certain aspects, method of the embodiments concern targetingof CD19 or HERV-K-expressing cells. For example, a HERV-K targeted CARcell can comprise a CAR including the scFv sequence of monoclonalantibody 6H5. In still further aspects, a CAR of the embodiments can beconjugated or fused with a cytokine, such as IL-2, IL-7, IL-15, IL-21 ora combination thereof.

In some embodiments, methods are provided for treating an individualwith a medical condition comprising the step of providing an effectiveamount of cells from the population of cells described herein, includingmore than once in some aspects, such as at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, or more days apart. In specific aspects, thecancer is cancer of the bladder, blood, bone, bone marrow, brain,breast, colon, esophagus, gastrointestinal, gum, head, kidney, liver,lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue,or uterus. In certain aspects, the cancer is a lymphoma, leukemia,non-Hodgkin's lymphoma, acute lymphoblastic leukemia, chroniclymphoblastic leukemia, chronic lymphocytic leukemia, or Bcell-associated autoimmune diseases.

As used herein in the specification and claims, “a” or “an” may mean oneor more. As used herein in the specification and claims, when used inconjunction with the word “comprising”, the words “a” or “an” may meanone or more than one. As used herein, in the specification and claim,“another” or “a further” may mean at least a second or more.

As used herein in the specification and claims, the term “about” is usedto indicate that a value includes the inherent variation of error forthe device, the method being employed to determine the value, or thevariation that exists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating certain embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: An exemplary strategy for transposase-based engineering ofcells. In this example, engineered T-cells are produced, which express aCAR. T cells are co-transfected with a transposon DNA construct encodinga CAR flanked by transposon repeats along with a mRNA encoding thetransposase. In this case, a 4D-NUCLEOFECTOR™ electroporation system(Lonza Group Ltd., Switzerland) is used for the transfection. Onceintroduced into the cells, the transposase is transiently expressed andmediates integration of the CAR construct into genomic DNA. The mRNAencoding the transposase is degraded within the cells, ensuring nolog-term expression of transposase in the cells.

FIG. 2: Histograms show flow cytometry data of human T-cells transfectedwith CAR as outlined in FIG. 1. Results are compared between tworecombinant transposases of the embodiments, hSB110 and hSB81, versusSB100x. Upper panels show the number of cells positive for CAR (y-axis)versus cells that have been rendered non-viable, as assessed by 7AAD(7-Aminoactinomycin D) staining (x-axis), 8-days post transfection.Lower panels show the number of cells positive for CAR expression(y-axis) and which express CD3 (x-axis), 15-days post transfection.Results of the studies show that the recombinant transposases of theembodiments are significantly more efficient at engineering cells thanSB100x. By day 8 post transfection, over 22% of the cells electroporatedwith hSB110 and hSB81 coding mRNAs express CAR as compared to only 15%of cells resulting from the use of SB100x. Likewise, by day 15, over 80%of the cell population electroporated with hSB110 and hSB81 co-expressedCAR and CD3 as compared to only 73.4% of cells resulting from the use ofSB100x.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. Genetic Engineering of Cells

Genetic engineering of cells has emerged as a powerful technique forproviding stable expression of desired genes in a wide range of cells.Recently, such technology has even been applied to cells used fortherapeutic intervention in a range of disease conditions. For example,genetically engineered T-cells that express receptors targeted to adisease-associated antigen are currently in clinical trials as ananti-cancer therapy. Transposase systems are a desirable system for usein engineering, especially in the case of cells used as therapeutics,since they do not introduce heterologous genetic elements that aremaintained in the cells, a common feature of virus-based engineeringsystems. However, high efficiency is needed to provide a sufficientnumber of engineered T-cells that are required for therapeuticintervention.

Studies herein demonstrate new recombinant transposase enzymes, termedhSB110 and hSB81, which exhibit significantly improved efficiency inengineering of human cells. For example, when used for integration ofCAR expression constructs into primary human T-cells, the newtransposases were able to produce populations of cells where well over20% of the cells exhibited CAR expression by day 8 post transfection(FIG. 2). Moreover, by day 15, over 80% of the cells in the transfectedpopulation were positive for CAR expression (FIG. 2). Such highefficiency genetic engineering of human cells represents a significantimprovement over previous transposase-based engineering systems.

Transposase coding sequences of the embodiments can be employed for highefficiency genetic engineering of mammalian cells. For example, thetransposases can be used for rapid production of populations ofCAR-expressing T-cells. In an embodiment, mRNA encoding the transposaseis co-transfected into T-cells (or T-cell precursors) along with a DNAvector encoding the desired CAR expression cassette, flanked bytransposon repeats. The CAR-expressing T-cells may then be purifiedand/or selectively expanded. In the case of T-cells used for therapy, itis desirable that as little expansion as possible be needed in order toreduce the time and cost of cell preparation. Importantly, thetransposases provided herein significantly improve such an engineeringprocedure by increasing the proportion of cells that exhibit stableexpression of the CAR and, which are therefore available for furtherexpansion.

II. Transposase Polypeptides and Coding Sequences

As described in the foregoing summary, certain aspects of theembodiments concern recombinant transposase polypeptides and nucleicacids encoding the same. In certain aspects, a transposases for useaccording to the embodiments is a polypeptide comprising or consistingof a sequence at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1or SEQ ID NO: 3. In certain aspects, the transposase embodiments maycomprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions,deletions or insertions relative to the sequences of SEQ ID NO: 1 or SEQID NO: 3. For example, transposase polypeptides of the embodiments maybe further modified by one or more amino acid substitutions whilemaintaining their enzymatic activity. In some cases, an amino acidposition can be substituted for an amino acid at a correspondingposition of a different transposase sequence. For example, the sequenceof additional transposases are provided in U.S. Pat. Nos. 6,489,458;7,148,203; 8,227,432; U.S. Patent Publn. No. 2011/0117072; Mates et al.,2009 and in Ivics et al., 1997, each of which are incorporated herein byreference in their entirety.

In some aspects, amino acid substitutions can be made at one or morepositions wherein the substitution is for an amino acid having a similarhydrophilicity. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like. Thussuch conservative substitution can be made in a transposase and willlikely only have minor effects on their activity. As detailed in U.S.Pat. No. 4,554,101, the following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline(−0.5±1); alanine (0.5); histidine −0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). These values can beused as a guide and thus substitution of amino acids whosehydrophilicity values are within ±2, those that are within ±1, and thosewithin ±0.5 comprise contemplated embodiments. Thus, any of thetransposase polypeptides described herein may be modified by thesubstitution of an amino acid, for different, but homologous amino acidwith a similar hydrophilicity value. Amino acids with hydrophilicitieswithin +/−1.0, or +/−0.5 points are considered homologous.

In still further aspects, a transposase polypeptide of the embodimentsis fused to a heterologous polypeptide sequence, such as a purificationtag (e.g., a T7, poly-His or GST tag), a reporter or a CPP. For examplethe polypeptide may be fused (or conjugated) to a reporter, such as animaging agent. It will be understood that in certain cases, a fusionprotein may comprise additional amino acids positioned between thetransposase and a heterologous polypeptide. In general these sequencesare interchangeably termed “linker sequences” or “linker regions.” Oneof skill in the art will recognize that linker regions may be one ormore amino acids in length and often comprise one or more glycineresidue(s) which confer flexibility to the linker. Such linker sequencescan be repeated 1, 2, 3, 4, 5, 6, or more times or combined with one ormore different linkers to form an array of linker sequences. Forinstance, in some applications, a linker region may comprise a proteasecleavage site (e.g., for the removal of a purification tag or CPP).

As used herein the terms CPP and membrane translocation peptide (MTP) asused interchangeably to refer to peptide sequences that enhance theability of a polypeptide to be internalized by a cell. Examples for CPPsfor use according to the embodiments include, without limitation,peptide segments derived from HIV Tat, herpes virus VP22, the DrosophilaAntennapedia homeobox gene product, protegrin I, the T1 CPP, the T2 CPP,or the INF7 CPP (see, e.g., U.S. Patent Pub. No. 20140140976,incorporated herein by reference).

III. Cell Engineering

Aspects of the embodiments concern genetic engineering of mammaliancells using transposases of the embodiments. Generally, such methodswill involve introducing into cells (i) a first vector encoding thetransposase (or a transposase polypeptide) and (ii) a second vectorencoding a desired genetic element that is flanked by transposonrepeats. Any type of mammalian cell can be genetically engineered bysuch a method. However, in certain aspects, the cell (cell population)is a stem cell, iPS cell, immune cell or a precursor of these cells.Methods described below address the specific example of T-cell (or otherimmune cell) engineering for CAR expression. A skilled artisan will,however, recognize that the methodologies could be equally applied toany given cell type or engineering construct.

Thus, in certain embodiments methods are provided for making and/orexpanding the antigen-specific redirected T cells that comprisestransfecting T cells with an expression vector containing a DNAconstruct encoding a CAR. Optionally, such cells are stimulated withantigen positive cells, recombinant antigen, or an antibody to thereceptor to cause the cells to proliferate.

In another aspect, a method is provided of stably transfecting andre-directing T cells. Such a transfection according to the embodimentscan be by any the various transfection techniques that are well known inthe art. In some aspects, nucleic acids can be introduced into cellsusing viral vectors or viral particles. Indeed, most investigators haveused viral vectors to carry heterologous genes into T cells. However, insome aspects, transfection of the embodiments does not involve the useof a viral vector. For instance, transfection can be by electroporation,use of charged or uncharged lipids, cationic polymers or polypeptides,salt precipitation or other non-viral nucleic acid transfer (such as,but not limited to sonoporation). In certain aspects, the transfectionuses naked DNA (or RNA or protein, in the case of a transposase). Byusing naked DNA, the time required to produce redirected T cells can bereduced. “Naked DNA” means DNA encoding a CAR is contained in anexpression cassette or vector in proper orientation for expression. Anelectroporation method of embodiments produces stable transfectants thatexpress and carry on their surfaces CAR.

In some aspects, a CAR of the embodiments can be further defined as a“chimeric TCR” means a receptor that is expressed by T cells and thatcomprises intracellular signaling, transmembrane, and extracellulardomains, where the extracellular domain is capable of specificallybinding in an MHC unrestricted manner an antigen that is not normallybound by a T-cell receptor in that manner. Stimulation of the T cells bythe antigen under proper conditions results in proliferation (expansion)of the cells and/or production of IL-2. The method is applicable totransfection with chimeric TCRs that are specific for any given targetantigens, such as chimeric TCRs that are specific for HER2/Neu(Stancovski et al., 1993), ERBB2 (Moritz et al., 1994), folate bindingprotein (Hwu et al., 1995), renal cell carcinoma (Weitjens et al.,1996), and HIV-1 envelope glycoproteins gp120 and gp41 (Roberts et al.,1994). Other cell-surface target antigens include, but are not limitedto, CD20, carcinoembryonic antigen, mesothelin, ROR1, c-Met, CD56, GD2,GD3, alphafetoprotein, CD23, CD30, CD123, IL-11Ralpha, kappa chain,lambda chain, CD70, CA-125, MUC-1, EGFR and variants, epithelial tumorantigen, and so forth.

In certain aspects, T cells for use according to the embodiments areprimary human T cells, such as T cells derived from human peripheralblood mononuclear cells (PBMC), PBMC collected after stimulation withG-CSF, bone marrow, or umbilical cord blood. Conditions include the useof mRNA and DNA and electroporation. Following transfection the cellsmay be immediately infused or may be stored. In certain aspects,following transfection, the cells may be propagated for days, weeks, ormonths ex vivo as a bulk population within about 1, 2, 3, 4, 5 days ormore following gene transfer into cells. In a further aspect, followingtransfection, the transfectants are cloned and a clone demonstratingpresence of a single integrated or episomally maintained expressioncassette or plasmid, and expression of the chimeric receptor is expandedex vivo. The clone selected for expansion demonstrates the capacity tospecifically recognize and lyse antigen-expressing target cells. Therecombinant T cells may be expanded by stimulation with IL-2, or othercytokines (e.g., IL-7, IL-12, IL-15, IL-21, and others). The recombinantT cells may be expanded by stimulation with artificial antigenpresenting cells. The recombinant T cells may be expanded on artificialantigen presenting cell or with an antibody, such as OKT3, which crosslinks CD3 on the T cell surface. Subsets of the recombinant T cells maybe deleted on artificial antigen presenting cell or with an antibody,such as alemtuzumab, which binds CD52 on the T cell surface. In afurther aspect, the genetically modified cells may be cryopreserved.

T-cell propagation (survival) after infusion may be assessed by: (i)q-PCR and/or digital PCR (e.g., Droplet Digital™ PCR (Bio-Rad, Hercules,Calif.) using primers specific for the transposon and/or CAR; (ii) flowcytometry using an antibody specific for the CAR; and/or (iii) flowcytometry using soluble TAA.

In certain embodiments of the invention, the CAR cells are delivered toan individual in need thereof, such as an individual that has cancer oran infection. The cells then enhance the individual's immune system toattack the respective cancer or pathogenic cells. In some cases, theindividual is provided with one or more doses of the antigen-specificCAR T-cells. In cases where the individual is provided with two or moredoses of the antigen-specific CAR T-cells, the duration between theadministrations should be sufficient to allow time for propagation inthe individual, and in specific embodiments the duration between dosesis 1, 2, 3, 4, 5, 6, 7, or more days.

A source of allogeneic or autologous T cells that are modified toinclude both a chimeric antigen receptor (and, in some cases, that lackfunctional TCR) may be of any kind, but in specific embodiments thecells are obtained from a bank of umbilical cord blood, peripheralblood, human embryonic stem cells, or induced pluripotent stem cells,for example. Suitable doses for a therapeutic effect would be at least10⁵ or between about 10⁵ and about 10¹⁰ cells per dose, for example,preferably in a series of dosing cycles. An exemplary dosing regimenconsists of four one-week dosing cycles of escalating doses, starting atleast at about 10⁵ cells on Day 0, for example increasing incrementallyup to a target dose of about 10¹⁰ cells within several weeks ofinitiating an intra-patient dose escalation scheme. Suitable modes ofadministration include intravenous, subcutaneous, intracavitary (forexample by reservoir-access device), intraperitoneal, and directinjection into a tumor mass.

A pharmaceutical composition of the present invention can be used aloneor in combination with other well-established agents useful for treatingcancer. Whether delivered alone or in combination with other agents, thepharmaceutical composition of the present invention can be delivered viavarious routes and to various sites in a mammalian, particularly human,body to achieve a particular effect. One skilled in the art willrecognize that, although more than one route can be used foradministration, a particular route can provide a more immediate and moreeffective reaction than another route. For example, intradermal deliverymay be advantageously used over inhalation for the treatment ofmelanoma. Local or systemic delivery can be accomplished byadministration comprising application or instillation of the formulationinto body cavities, inhalation or insufflation of an aerosol, or byparenteral introduction, comprising intramuscular, intravenous,intraportal, intrahepatic, peritoneal, subcutaneous, or intradermaladministration.

A composition of the embodiments can be provided in unit dosage formwherein each dosage unit, e.g., an injection, contains a predeterminedamount of the composition, alone or in appropriate combination withother active agents. The term unit dosage form as used herein refers tophysically discrete units suitable as unitary dosages for human andanimal subjects, each unit containing a predetermined quantity of thecomposition of the present invention, alone or in combination with otheractive agents, calculated in an amount sufficient to produce the desiredeffect, in association with a pharmaceutically acceptable diluent,carrier, or vehicle, where appropriate. The specifications for the unitdosage forms of the present invention depend on the particularpharmacodynamics associated with the pharmaceutical composition in theparticular subject.

Desirably an effective amount or sufficient number of the isolatedtransduced T-cells is present in the composition and introduced into thesubject such that long-term, specific, anti-tumor responses areestablished to reduce the size of a tumor or eliminate tumor growth orregrowth than would otherwise result in the absence of such treatment.Desirably, the amount of transduced T cells reintroduced into thesubject causes about or at least about 10%, about or at least about 20%,about or at least about 30%, about or at least about 40%, about or atleast about 50%, about or at least about 60%, about or at least about70%, about or at least about 80%, about or at least about 90%, about orat least about 95%, about or at least about 98%, or about or a 100%decrease in tumor size when compared to the original or initial (e.g.,“therapy day 0”) size of the tumor.

Accordingly, the amount of transduced T cells administered should takeinto account the route of administration and should be such that asufficient number of the transduced T cells will be introduced so as toachieve the desired therapeutic response. Furthermore, the amounts ofeach active agent included in the compositions described herein (e.g.,the amount per each cell to be contacted or the amount per certain bodyweight) can vary in different applications. In general, theconcentration of transduced T cells desirably should be sufficient toprovide in the subject being treated at least from about 1×10⁶ to about1×10⁹ transduced T cells, even more desirably, from about 1×10⁷ to about5×10⁸ transduced T cells, although any suitable amount can be utilizedeither above, e.g., greater than 5×10⁸ cells, or below, e.g., less than1×10′ cells. The dosing schedule can be based on well-establishedcell-based therapies (see, e.g., Topalian and Rosenberg, 1987; U.S. Pat.No. 4,690,915), or an alternate continuous infusion strategy can beemployed.

These values provide general guidance of the range of transduced T cellsto be utilized by the practitioner upon optimizing the method of thepresent invention for practice of the invention. The recitation hereinof such ranges by no means precludes the use of a higher or lower amountof a component, as might be warranted in a particular application. Forexample, the actual dose and schedule can vary depending on whether thecompositions are administered in combination with other pharmaceuticalcompositions, or depending on interindividual differences inpharmacokinetics, drug disposition, and metabolism. One skilled in theart readily can make any necessary adjustments in accordance with theexigencies of the particular situation.

IV. Engineering Constructs

In certain specific aspects, a transposase system of the embodiments isused to engineer a cell with an expression construct ending a selectedgenetic element. In such aspects, the selected genetic element isflanked by transposon repeats that are functional with a transposase ofthe embodiments, such as the IR/DR sequences. The selected geneticelement may comprise any sequence desired to be transfected into a cell,but in certain aspects the element encodes a polypeptide coding sequenceand appropriate expression control sequences for mammalian expression.In some specific aspects, the selected genetic element encodes anantigen binding moiety, such as an antibody, a T-cell receptor or achimeric antigen receptor (CAR). As used herein, the term “antigen” is amolecule capable of being bound by an antibody or T-cell receptor orCAR.

Thus, embodiments of the present invention involve nucleic acids,including nucleic acids encoding an antigen-specific CAR polypeptide,including a CAR that has been humanized to reduce immunogenicity (hCAR),comprising an intracellular signaling domain, a transmembrane domain,and an extracellular domain comprising one or more signaling motifs. Incertain embodiments, the CAR may recognize an epitope comprised of theshared space between one or more antigens. Pattern recognitionreceptors, such as Dectin-1, may be used to derive specificity to acarbohydrate antigen. In certain embodiments, the binding region cancomprise complementary determining regions of a monoclonal antibody,variable regions of a monoclonal antibody, and/or antigen bindingfragments thereof. In another embodiment, that specificity is derivedfrom a peptide (e.g., cytokine) that binds to a receptor. Acomplementarity determining region (CDR) is a short amino acid sequencefound in the variable domains of antigen receptor (e.g., immunoglobulinand T-cell receptor) proteins that complements an antigen and thereforeprovides the receptor with its specificity for that particular antigen.Each polypeptide chain of an antigen receptor contains three CDRs (CDR1,CDR2, and CDR3). Since the antigen receptors are typically composed oftwo polypeptide chains, there are six CDRs for each antigen receptorthat can come into contact with the antigen—each heavy and light chaincontains three CDRs. Because most sequence variation associated withimmunoglobulins and T-cell receptors are found in the CDRs, theseregions are sometimes referred to as hypervariable domains. Among these,CDR3 shows the greatest variability as it is encoded by a recombinationof the VJ (VDJ in the case of heavy chain and TCR αβ chain) regions.

It is contemplated that the human CAR nucleic acids are human genes toenhance cellular immunotherapy for human patients. In a specificembodiment, the invention includes a full length CAR cDNA or codingregion. The antigen binding regions or domain can comprise a fragment ofthe VH and VL chains of a single-chain variable fragment (scFv) derivedfrom a particular human monoclonal antibody, such as those described inU.S. Pat. No. 7,109,304, incorporated herein by reference or it cancomprise any other antigen-binding moiety. The fragment can also be anynumber of different antigen binding domains of a human antigen-specificantibody. In a more specific embodiment, the fragment is anantigen-specific scFv encoded by a sequence that is optimized for humancodon usage for expression in human cells.

The arrangement could be multimeric, such as a diabody or multimers. Themultimers are most likely formed by cross pairing of the variableportion of the light and heavy chains into what has been referred to byWinters as a diabody. The hinge portion of the construct can havemultiple alternatives from being totally deleted, to having the firstcysteine maintained, to a proline rather than a serine substitution, tobeing truncated up to the first cysteine. The Fc portion can be deleted.Any protein that is stable and/or dimerizes can serve this purpose. Onecould use just one of the Fc domains, e.g., either the CH2 or CH3 domainfrom human immunoglobulin. One could also use the hinge, CH2 and CH3region of a human immunoglobulin that has been modified to improvedimerization. One could also use just the hinge portion of animmunoglobulin. One could also use portions of CD8alpha.

The intracellular signaling domain of a chimeric antigen receptor of theembodiments is responsible for activation of at least one of the normaleffector functions of the immune cell in which the chimeric receptor hasbeen placed. The term “effector function” refers to a specializedfunction of a differentiated cell. Effector function of a T cell, forexample, may be cytolytic activity or helper activity including thesecretion of cytokines. Effector function in a naive, memory, ormemory-type T cell includes antigen-dependent proliferation. Thus theterm “intracellular signaling domain” refers to the portion of a proteinthat transduces the effector function signal and directs the cell toperform a specialized function. While usually the entire intracellularsignaling domain will be employed, in many cases it will not benecessary to use the entire intracellular polypeptide. To the extentthat a truncated portion of the intracellular signaling domain may finduse, such truncated portion may be used in place of the intact chain aslong as it still transduces the effector function signal. The termintracellular signaling domain is thus meant to include any truncatedportion of the intracellular signaling domain sufficient to transducethe effector function signal. Examples include the zeta chain of theT-cell receptor or any of its homologs (e.g., eta, delta, gamma, orepsilon), MB1 chain, B29, Fc RIII, Fc RI, and combinations of signalingmolecules, such as CD3ζ and CD28, CD27, 4-1BB, DAP-10, OX40, andcombinations thereof, as well as other similar molecules and fragments.Intracellular signaling portions of other members of the families ofactivating proteins can be used, such as FcγRIII and FcεRI. See Gross etal. (1992), Stancovski et al. (1993), Moritz et al. (1994), Hwu et al.(1995), Weijtens et al. (1996), and Hekele et al. (1996) for disclosuresof chimeric T cell receptors using these alternative transmembrane andintracellular domains. In certain embodiments, the human CD3ζintracellular domain is used for activation.

The antigen-specific extracellular domain and the intracellularsignaling-domain may be linked by a transmembrane domain, such as thehuman IgG4Fc hinge and Fc regions. Alternatives include the human CD4transmembrane domain, the human CD28 transmembrane domain, thetransmembrane human CD3ζ domain, or a cysteine mutated human CD3ζdomain, or other transmembrane domains from other human transmembranesignaling proteins, such as CD16 and CD8 and erythropoietin receptor.Additional modifications can be added to the transmembrane amino acidsequences.

In some embodiments, the CAR nucleic acid comprises a sequence encodingother costimulatory receptors, such as a transmembrane domain and amodified CD28 intracellular signaling domain. Other costimulatoryreceptors include, but are not limited to one or more of CD28, CD27,OX-40 (CD134), DAP10, and 4-1BB (CD137). In addition to a primary signalinitiated by CD3ζ, an additional signal provided by a humancostimulatory receptor inserted in a human CAR is important for fullactivation of T cells and could help improve in vivo persistence and thetherapeutic success of the adoptive immunotherapy.

In particular embodiments, the invention concerns isolated nucleic acidsegments and expression cassettes incorporating DNA sequences thatencode the CAR. Vectors of the present invention are designed,primarily, to deliver desired genes to immune cells, preferably T cellsunder the control of regulated eukaryotic promoters, for example, MNDU3promoter, CMV promoter, EFlalpha promoter, or Ubiquitin promoter. Also,the vectors may contain a selectable marker, if for no other reason, tofacilitate their manipulation in vitro. In other embodiments, the CARcan be expressed from mRNA in vitro transcribed from a DNA template.

Chimeric antigen receptor molecules are recombinant and aredistinguished by their ability to both bind antigen and transduceactivation signals via immunoreceptor activation motifs (ITAM's) presentin their cytoplasmic tails. Receptor constructs utilizing anantigen-binding moiety (for example, generated from single chainantibodies (scFv)) afford the additional advantage of being “universal”in that they bind native antigen on the target cell surface in anHLA-independent fashion. For example, several laboratories have reportedon scFv constructs fused to sequences coding for the intracellularportion of the CD3 complex's zeta chain (ζ), the Fc receptor gammachain, and sky tyrosine kinase (Eshhar et al., 1993; Fitzer-Attas etal., 1998). Re-directed T cell effector mechanisms including tumorrecognition and lysis by CTL have been documented in several murine andhuman antigen-scFv: ζ systems (Eshhar, 1997; Altenschmidt et al., 1997;Brocker et al., 1998).

To date non-human antigen binding regions are typically used inconstructing a chimeric antigen receptor. A potential problem with usingnon-human antigen binding regions, such as murine monoclonal antibodies,is the lack of human effector functionality and inability to penetrateinto tumor masses. In other words, such antibodies may be unable tomediate complement-dependent lysis or lyse human target cells throughantibody-dependent cellular toxicity or Fc-receptor mediatedphagocytosis to destroy cells expressing CAR. Furthermore, non-humanmonoclonal antibodies can be recognized by the human host as a foreignprotein, and therefore, repeated injections of such foreign antibodiescan lead to the induction of immune responses leading to harmfulhypersensitivity reactions. For murine-based monoclonal antibodies, thisis often referred to as a Human Anti-Mouse Antibody (HAMA) response.Therefore, the use of human antibodies is more preferred because they donot elicit as strong a HAMA response as murine antibodies. Similarly,the use of human sequences in the CAR can avoid immune-mediatedrecognition and therefore elimination by endogenous T cells that residein the recipient and recognize processed antigen in the context of HLA.

In some embodiments, the chimeric antigen receptor comprises: a) anintracellular signaling domain, b) a transmembrane domain, and c) anextracellular domain comprising an antigen binding region.

In specific embodiments, intracellular receptor signaling domains in theCAR include those of the T cell antigen receptor complex, such as thezeta chain of CD3, also Fcγ RIII costimulatory signaling domains, CD28,CD27, DAP10, CD137, OX40, CD2, alone or in a series with CD3zeta, forexample. In specific embodiments, the intracellular domain (which may bereferred to as the cytoplasmic domain) comprises part or all of one ormore of TCR zeta chain, CD28, CD27, OX40/CD134, 4-1BB/CD137, FcεRIγ,ICOS/CD278, IL-2Rbeta/CD122, IL-2Ralpha/CD132, DAP10, DAP12, and CD40.In some embodiments, one employs any part of the endogenous T cellreceptor complex in the intracellular domain. One or multiplecytoplasmic domains may be employed, as so-called third generation CARshave at least two or three signaling domains fused together for additiveor synergistic effect, for example.

In certain embodiments of the chimeric antigen receptor, theantigen-specific portion of the receptor (which may be referred to as anextracellular domain comprising an antigen binding region) comprises atumor associated antigen or a pathogen-specific antigen binding domainincluding carbohydrate antigen recognized by pattern-recognitionreceptors, such as Dectin-1. A tumor associated antigen may be of anykind so long as it is expressed on the cell surface of tumor cells.Exemplary embodiments of tumor associated antigens include CD19, CD20,carcinoembryonic antigen, alphafetoprotein, CA-125, MUC-1, CD56, EGFR,c-Met, AKT, Her2, Her3, epithelial tumor antigen, melanoma-associatedantigen, mutated p53, mutated ras, and so forth. In certain embodiments,the CAR can be co-expressed with a membrane-bound cytokine to improvepersistence when there is a low amount of tumor-associated antigen. Forexample, CAR can be co-expressed with membrane-bound IL-15.

In certain embodiments intracellular tumor associated antigens may betargeted, such as HA-1, survivin, WT1, and p53. This can be achieved bya CAR expressed on a universal T cell that recognizes the processedpeptide described from the intracellular tumor associated antigen in thecontext of HLA. In addition, the universal T cell may be geneticallymodified to express a T-cell receptor pairing that recognizes theintracellular processed tumor associated antigen in the context of HLA.

The pathogen may be of any kind, but in specific embodiments thepathogen is a fungus, bacteria, or virus, for example. Exemplary viralpathogens include those of the families of Adenoviridae, Epstein-Barrvirus (EBV), Cytomegalovirus (CMV), Respiratory Syncytial Virus (RSV),JC virus, BK virus, HSV, HHV family of viruses, Picornaviridae,Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae,Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus,Rhabdoviridae, and Togaviridae. Exemplary pathogenic viruses causesmallpox, influenza, mumps, measles, chickenpox, ebola, and rubella.Exemplary pathogenic fungi include Candida, Aspergillus, Cryptococcus,Histoplasma, Pneumocystis, and Stachybotrys. Exemplary pathogenicbacteria include Streptococcus, Pseudomonas, Shigella, Campylobacter,Staphylococcus, Helicobacter, E. coli, Rickettsia, Bacillus, Bordetella,Chlamydia, Spirochetes, and Salmonella. In one embodiment the pathogenreceptor Dectin-1 can be used to generate a CAR that recognizes thecarbohydrate structure on the cell wall of fungi. T cells geneticallymodified to express the CAR based on the specificity of Dectin-1 canrecognize Aspergillus and target hyphal growth. In another embodiment,CARs can be made based on an antibody recognizing viral determinants(e.g., the glycoproteins from CMV and Ebola) to interrupt viralinfections and pathology.

In some embodiments, the pathogenic antigen is an Aspergilluscarbohydrate antigen for which the extracellular domain in the CARrecognizes patterns of carbohydrates of the fungal cell wall, such asvia Dectin-1.

A chimeric immunoreceptor according to the present invention can beproduced by any means known in the art, though preferably it is producedusing recombinant DNA techniques. A nucleic acid sequence encoding theseveral regions of the chimeric receptor can be prepared and assembledinto a complete coding sequence by standard techniques of molecularcloning (genomic library screening, PCR, primer-assisted ligation, scFvlibraries from yeast and bacteria, site-directed mutagenesis, etc.). Theresulting coding region can be inserted into an expression vector andused to transform a suitable expression host allogeneic T-cell line.

As used herein, a nucleic acid construct or nucleic acid sequence orpolynucleotide is intended to mean a DNA molecule that can betransformed or introduced into a T cell and be transcribed andtranslated to produce a product (e.g., a chimeric antigen receptor).

In an exemplary nucleic acid construct (polynucleotide) employed in thepresent embodiments, the promoter is operably linked to the nucleic acidsequence encoding the chimeric receptor, i.e., they are positioned so asto promote transcription of the messenger RNA from the DNA encoding thechimeric receptor. The promoter can be of genomic origin orsynthetically generated. A variety of promoters for use in T cells arewell-known in the art (e.g., the CD4 promoter disclosed by Marodon etal. (2003)). The promoter can be constitutive or inducible, whereinduction is associated with the specific cell type or a specific levelof maturation, for example. Alternatively, a number of well-known viralpromoters are also suitable. Promoters of interest include the β-actinpromoter, SV40 early and late promoters, immunoglobulin promoter, humancytomegalovirus promoter, retrovirus promoter, and the Friend spleenfocus-forming virus promoter. The promoters may or may not be associatedwith enhancers, wherein the enhancers may be naturally associated withthe particular promoter or associated with a different promoter.

The sequence of the open reading frame encoding the chimeric receptorcan be obtained from a genomic DNA source, a cDNA source, or can besynthesized (e.g., via PCR), or combinations thereof. Depending upon thesize of the genomic DNA and the number of introns, it may be desirableto use cDNA or a combination thereof as it is found that intronsstabilize the mRNA or provide T cell-specific expression (Barthel andGoldfeld, 2003). Also, it may be further advantageous to use endogenousor exogenous non-coding regions to stabilize the mRNA.

For expression of a chimeric antigen receptor of the present invention,the naturally occurring or endogenous transcriptional initiation regionof the nucleic acid sequence encoding N-terminal components of thechimeric receptor can be used to generate the chimeric receptor in thetarget host. Alternatively, an exogenous transcriptional initiationregion can be used that allows for constitutive or inducible expression,wherein expression can be controlled depending upon the target host, thelevel of expression desired, the nature of the target host, and thelike.

Likewise, a signal sequence directing the chimeric receptor to thesurface membrane can be the endogenous signal sequence of N-terminalcomponent of the chimeric receptor. Optionally, in some instances, itmay be desirable to exchange this sequence for a different signalsequence. However, the signal sequence selected should be compatiblewith the secretory pathway of T cells so that the chimeric receptor ispresented on the surface of the T cell.

Similarly, a termination region may be provided by the naturallyoccurring or endogenous transcriptional termination region of thenucleic acid sequence encoding the C-terminal component of the chimericreceptor. Alternatively, the termination region may be derived from adifferent source. For the most part, the source of the terminationregion is generally not considered to be critical to the expression of arecombinant protein and a wide variety of termination regions can beemployed without adversely affecting expression.

The chimeric constructs of the present invention find application insubjects having or suspected of having cancer by reducing the size of atumor or preventing the growth or re-growth of a tumor in thesesubjects. Accordingly, the present invention further relates to a methodfor reducing growth or preventing tumor formation in a subject byintroducing a chimeric construct of the present invention into anisolated T cell of the subject and reintroducing into the subject thetransformed T cell, thereby effecting anti-tumor responses to reduce oreliminate tumors in the subject. Suitable T cells that can be usedinclude cytotoxic lymphocytes (CTL) or any cell having a T cell receptorin need of disruption. As is well-known to one of skill in the art,various methods are readily available for isolating these cells from asubject. For example, using cell surface marker expression or usingcommercially available kits (e.g., ISOCELL™ from Pierce, Rockford,Ill.).

It is contemplated that the chimeric construct can be introduced intothe subject's own T cells as naked DNA, combined with other reagents(including but not limited to lipids, cationic polymers, PEG-complexes,protein complexes), or in a suitable vector. Methods of stablytransfecting T cells by electroporation using naked DNA are known in theart. See, e.g., U.S. Pat. No. 6,410,319, incorporated herein byreference. Naked DNA generally refers to the DNA encoding a chimericreceptor of the present invention contained in a plasmid expressionvector in proper orientation for expression. Advantageously, the use ofnaked DNA reduces the time required to produce T cells expressing thechimeric receptor of the present invention.

Once it is established that the transfected or transduced T cell iscapable of expressing the chimeric receptor as a surface membraneprotein with the desired regulation and at a desired level, it can bedetermined whether the chimeric receptor is functional in the host cellto provide for the desired signal induction. Subsequently, thetransduced T cells are reintroduced or administered to the subject toactivate anti-tumor responses in the subject. To facilitateadministration, the transduced T cells according to the invention can bemade into a pharmaceutical composition or made into an implantappropriate for administration in vivo, with appropriate carriers ordiluents, which further can be pharmaceutically acceptable. The means ofmaking such a composition or an implant have been described in the art(see, for instance, Remington's Pharmaceutical Sciences, 16th Ed., Mack,ed. (1980)). Where appropriate, the transduced T cells can be formulatedinto a preparation in semisolid or liquid form, such as a capsule,solution, injection, inhalant, or aerosol, in the usual ways for theirrespective route of administration. Means known in the art can beutilized to prevent or minimize release and absorption of thecomposition until it reaches the target tissue or organ, or to ensuretimed-release of the composition. Desirably, however, a pharmaceuticallyacceptable form is employed that does not ineffectuate the cellsexpressing the chimeric receptor. Thus, desirably the transduced T cellscan be made into a pharmaceutical composition containing a balanced saltsolution, preferably Hanks' balanced salt solution, or normal saline.

V. Kits of the Embodiments

Any of the compositions described herein may be comprised in a kit. Insome aspects, a transposases polypeptide of the embodiments, or anucleic acid encoding the same, is provided in the kit. Such a kit mayinclude a variety of additional elements, such a DNA vector encodingtransposon repeats, transfection reagents, cells, a CAR expressionconstruct, media, aAPCs, growth factors, antibodies (e.g., for sortingor characterizing CAR T-cells) and/or plasmids encoding CARs ortransposase.

In a non-limiting example, a kit comprises a transposases polypeptide ofthe embodiments, or a nucleic acid encoding the same, one or morereagents to generate a CAR expression construct (having flankingtransposon repeats), cells for transfection of the expression construct,and/or one or more instruments to obtain cells for transfection of theexpression construct (such an instrument may be a syringe, pipette,forceps, and/or any such medically approved apparatus). In still afurther aspects, a transfection device such as an electroporation deviceis included.

In some embodiments, an expression construct for eliminating endogenousTCR α/β expression, one or more reagents to generate the construct,and/or CAR+ T cells are provided in the kit. In some embodiments, thereincludes expression constructs that encode zinc finger nuclease(s).

The kits may comprise one or more suitably aliquoted compositions of thepresent invention or reagents to generate compositions of the invention.The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits may include at leastone vial, test tube, flask, bottle, syringe, or other container means,into which a component may be placed, and preferably, suitablyaliquoted. Where there is more than one component in the kit, the kitalso will generally contain a second, third, or other additionalcontainer into which the additional components may be separately placed.However, various combinations of components may be comprised in a vial.The kits of the present invention also will typically include a meansfor containing the chimeric receptor construct and any other reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow molded plastic containers into which thedesired vials are retained, for example.

VI. EXAMPLES

The following examples are included to demonstrate certain embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—Recombinant Transposases with High Activity in Human Cells

DNA sequences encoding transposases originally derived from Salmo salar(Atlantic salmon) were engineered and humanized in an attempt to produceenzymes with increased efficiency in human cells. The sequences of twoof the produced transposases (and their nucleic acid coding sequences)are shown below and have been named as hSB110 and hSB81.

hSB110 (SEQ ID NO: 1): 1mgkskeisqd lrkrivdlhk sgsslgaisk rlavprssvq tivrkykhhg ttqpsyrsgr 61rrvlsprder tlvrkvqinp rttakdlvkm leetgtkvsi stvkrvlyrh nlkghsarkk 121pllqnrhkka rlrfarahgd kdrtfwrnvl wsdetkielf ghndhryvwr kkgeackpkn 181tiptvkhggg simlwgcfaa ggtgalhkid gimdavqyvd ilkqhlktsv rklklgrkwv 241fqhdndpkht skhvrkwlkd nkvkvlewps qspdlnpien lwaelkkrvr arrptnltql 301hqlcqeewak ihpnycgklv egypkrltqv kqfkgnatky hSB110 (SEQ ID NO: 2): 1ATGGGCAAGA GCAAAGAGAT CAGCCAGGAC CTGCGGAAGC GGATCGTGGA CCTGCACAAG 61AGCGGCTCTA GCCTGGGCGC CATCAGCAAG AGACTGGCCG TGCCTAGAAG CAGCGTGCAG 121ACCATCGTGC GGAAGTACAA GCACCACGGC ACCACCCAGC CCAGCTACAG ATCTGGAAGG 181CGGAGAGTGC TGAGCCCCAG GGACGAGAGA ACACTCGTGC GCAAGGTGCA GATCAACCCC 241CGGACCACCG CCAAGGACCT CGTGAAGATG CTGGAAGAGA CAGGCACCAA GGTGTCCATC 301AGCACCGTGA AGCGGGTGCT GTACCGGCAC AACCTGAAGG GCCACAGCGC CAGAAAGAAG 361CCCCTGCTGC AGAACAGACA CAAGAAGGCC CGGCTGAGAT TCGCCAGAGC CCACGGCGAC 421AAGGACAGAA CCTTCTGGCG GAACGTGCTG TGGAGCGACG AGACAAAGAT CGAGCTGTTC 481GGCCACAACG ACCACAGATA CGTGTGGCGG AAGAAGGGCG AGGCCTGCAA GCCCAAGAAC 541ACCATCCCCA CAGTGAAGCA CGGCGGAGGC AGCATCATGC TGTGGGGCTG TTTTGCCGCT 601GGCGGCACAG GCGCCCTGCA CAAAATCGAC GGCATCATGG ACGCCGTGCA GTACGTGGAC 661ATCCTGAAGC AGCACCTGAA AACCTCTGTG CGGAAGCTGA AGCTGGGCCG GAAATGGGTG 721TTCCAGCACG ACAACGACCC CAAGCACACC AGCAAGCACG TGCGGAAATG GCTGAAGGAC 781AACAAAGTGA AAGTGCTGGA ATGGCCCAGC CAGTCCCCCG ACCTGAACCC CATCGAAAAC 841CTGTGGGCCG AGCTGAAGAA AAGAGTGCGG GCCAGACGGC CCACCAACCT GACACAGCTG 901CACCAGCTGT GCCAGGAAGA GTGGGCCAAG ATCCACCCCA ACTACTGCGG CAAGCTGGTG 961GAAGGCTACC CCAAGAGGCT GACCCAAGTG AAACAGTTCA AGGGCAACGC CACCAAGTAC 1021TGA hSB81 (SEQ ID NO: 3): 1mgkskeisqd lrkrivdlhk sgsslgaisk rlavprssvq tivrkykhhg ttqpsyrsgr 61rrvlsprder tlvrkvqinp rttakdlvkm leetgtkvsi stvkrvlyrh nlkghsarkk 121pllqnrhkka rlrfarahgd kdrtfwrnvl wsdetkielf ghndhryvwr kkgeackpkn 181tiptvkhggg simlwgcfaa ggtgalhkid gimdavqyvd ilkqhlktsv rklklgrkwv 241fqhdndpkht skhvrkwlkd nkvkvlewps qspdlnpien lwaelkkrvr arrptnltql 301hqlcqeewak ihptycgklv egypkrltqv kqfkgnatky hSB81 (SEQ ID NO: 4): 1ATGGGCAAGA GCAAAGAGAT CAGCCAGGAC CTGCGGAAGC GGATCGTGGA CCTGCACAAG 61AGCGGCTCTA GCCTGGGCGC CATCAGCAAG AGACTGGCCG TGCCTAGAAG CAGCGTGCAG 121ACCATCGTGC GGAAGTACAA GCACCACGGC ACCACCCAGC CCAGCTACAG ATCTGGAAGG 181CGGAGAGTGC TGAGCCCCAG GGACGAGAGA ACACTCGTGC GCAAGGTGCA GATCAACCCC 241CGGACCACCG CCAAGGACCT CGTGAAGATG CTGGAAGAGA CAGGCACCAA GGTGTCCATC 301AGCACCGTGA AGCGGGTGCT GTACCGGCAC AACCTGAAGG GCCACAGCGC CAGAAAGAAG 361CCCCTGCTGC AGAACAGACA CAAGAAGGCC CGGCTGAGAT TCGCCAGAGC CCACGGCGAC 421AAGGACAGAA CCTTCTGGCG GAACGTGCTG TGGAGCGACG AGACAAAGAT CGAGCTGTTC 481GGCCACAACG ACCACAGATA CGTGTGGCGG AAGAAGGGCG AGGCCTGCAA GCCCAAGAAC 541ACCATCCCCA CAGTGAAGCA CGGCGGAGGC AGCATCATGC TGTGGGGCTG TTTTGCCGCT 601GGCGGCACAG GCGCCCTGCA CAAAATCGAC GGCATCATGG ACGCCGTGCA GTACGTGGAC 661ATCCTGAAGC AGCACCTGAA AACCTCTGTG CGGAAGCTGA AGCTGGGCCG GAAATGGGTG 721TTCCAGCACG ACAACGACCC CAAGCACACC AGCAAGCACG TGCGGAAATG GCTGAAGGAC 781AACAAAGTGA AAGTGCTGGA ATGGCCCAGC CAGTCCCCCG ACCTGAACCC CATCGAAAAC 841CTGTGGGCCG AGCTGAAGAA AAGAGTGCGG GCCAGACGGC CCACCAACCT GACACAGCTG 901CACCAGCTGT GCCAGGAAGA GTGGGCCAAG ATCCACCCCA CCTACTGCGG CAAGCTGGTG 961GAAGGCTACC CCAAGAGGCT GACCCAAGTG AAACAGTTCA AGGGCAACGC CACCAAGTAC 1021TGA

The hSB110 and hSB81 transposases, as well as a control transposase,were then tested for their ability to produce engineered human T-cellswith a genetically integrated CAR. The protocol for these studies isshown in FIG. 1. Human T-cells were co-transfected with a transposon DNAconstruct encoding the CAR flanked by transposon repeats along with amRNA encoding the transposases polypeptides. For the tranfections a4D-NUCLEOFECTOR™ electroporation system (Lonza) was used. Followingelectroporation the cells were cultured and assessed for CAR expressionby flow cytometry.

The results of these studies are shown in FIG. 2. The histograms of theupper panels show the number of cells positive for CAR (y-axis) versuscells that have been rendered non-viable, as assessed by 7AAD staining(x-axis), 8-days post transfection. Lower panels show the number ofcells positive for CAR (y-axis) and which express CD3 (x-axis), 15-dayspost transfection. These results clearly demonstrate that the hSB110 andhSB81 transposases are significantly more efficient at engineering cellsthan SB100x. By day 8 post transfection, over 22% of the hSB110 andhSB81 electroporated cells express CAR. Only 15% of cells from theSB100x electroporation expressed CAR at this time point. No significantdifference was seen in the number of non-viable cells with any of thetest constructs (as assessed by 7AAD stain). Moreover, by day 15, over80% of the cell populations electroporated with hSB110 and hSB81co-expressed CAR and CD3.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of certain embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method of providing an immune response in ahuman subject in need thereof comprising: (a) transfecting an immunecell with (i) a nucleic acid encoding a recombinant polypeptide havingtransposase activity, wherein said polypeptide comprises an amino acidsequence at least 90% identical to the sequence of SEQ ID NO: 1, whereinthe polypeptide comprises: (i) an Arg at the position corresponding toposition 14; (ii) an Ala at the position corresponding to position 33;(iii) a His at the position corresponding to position 115; (iv) an Argat the position corresponding to position 136; (v) an Asp at theposition corresponding to position 214; (vi) an Ala at the positioncorresponding to position 215; (vii) a Val at the position correspondingto position 216; (viii) a Gln at the position corresponding to position217; (ix) a His at the position corresponding to position 243; (x) a Hisat the position corresponding to position 253; and (xi) an Arg at theposition corresponding to position 255; and (ii) a DNA encoding a CAR orTCR flanked by transposon repeats; and (b) administering the transfectedimmune cells expressing a CAR or TCR to the subject to provide a T-cellresponse.
 2. The method of claim 1, wherein the polypeptide comprises anamino acid sequence that is at least 90% identical to the sequence ofSEQ ID NO:
 1. 3. The method of claim 1, wherein the polypeptidecomprises an amino acid sequence that is at least 95% identical to thesequence of SEQ ID NO:
 1. 4. The method of claim 1, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:
 1. 5. Themethod of claim 1, wherein the polypeptide comprises an amino acidsequence that is at least 90% identical to the sequence of SEQ ID NO: 3.6. The method of claim 1, wherein the polypeptide comprises an aminoacid sequence that is at least 95% identical to the sequence of SEQ IDNO:
 3. 7. The method of claim 1, wherein the polypeptide comprises theamino acid sequence of SEQ ID NO:
 3. 8. The method of claim 1, whereinthe CAR specific to a tumor antigen.
 9. The method of claim 8, whereinthe tumor antigen is CD19, CD20, carcinoembryonic antigen,alpha-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen,melanoma-associated antigen, mutated p53, mutated ras, HER2/Neu, ERBB2,folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1envelope glycoprotein gp41, GD2, CD123, CD23, CD30, CD56, c-Met,mesothelin, GD3, HERV-K, IL-11Ralpha, kappa chain, lambda chain, CSPG4,ERBB2, EGFRvIII, or VEGFR2.
 10. The method of claim 1, furthercomprising transfecting the immune cells with a nucleic acid encoding amembrane-bound cytokine.
 11. The method of claim 10, wherein themembrane-bound cytokine is membrane-bound IL-15.
 12. The method of claim1, wherein the immune cell is a natural killer (NK) cell, a T-cell, aprecursor of a NK cell or a precursor of a T-cell.
 13. The method ofclaim 1, wherein the nucleic acid is mRNA.
 14. A method of treating ahuman subject having a disease cancer comprising: (a) transfecting animmune cell with (i) a nucleic acid encoding a recombinant polypeptidehaving transposase activity, wherein said polypeptide comprises an aminoacid sequence at least 90% identical to SEQ ID NO: 1, wherein thepolypeptide comprises: (i) an Arg at the position corresponding toposition 14; (ii) an Ala at the position corresponding to position 33;(iii) a His at the position corresponding to position 115; (iv) an Argat the position corresponding to position 136; (v) an Asp at theposition corresponding to position 214; (vi) an Ala at the positioncorresponding to position 215; (vii) a Val at the position correspondingto position 216; (viii) a Gln at the position corresponding to position217; (ix) a His at the position corresponding to position 243; (x) a Hisat the position corresponding to position 253; and (xi) an Arg at theposition corresponding to position 255; and (ii) a DNA encoding a CAR orTCR flanked by transposon repeats; and (b) administering atherapeutically effective amount of the transfected immune cellsexpressing a CAR or TCR to the subject, thereby effecting an anti-tumorresponse to reduce or eliminate tumors in the subject.
 15. The method ofclaim 14, wherein the cancer is cancer of the bladder, blood, bone, bonemarrow, brain, breast, colon, esophagus, gastrointestinal tract, gums,head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin,stomach, testis, tongue, or uterus.
 16. The method of claim 14, whereinthe cancer is a lymphoma, leukemia, non-Hodgkin's lymphoma, acutelymphoblastic leukemia, chronic lymphoblastic leukemia, chroniclymphocytic leukemia, or B cell-associated autoimmune disease.
 17. Themethod of claim 14, further comprising transfecting the immune cellswith a nucleic acid encoding a membrane-bound cytokine.
 18. The methodof claim 17, wherein the membrane-bound cytokine is membrane-boundIL-15.
 19. The method of claim 14, wherein the immune cell is a naturalkiller (NK) cell, a T-cell, a precursor of a NK cell or a precursor of aT-cell.